The complete mitochondrial genome of the freshwater crab Bottapotamon lingchuanense Türkay and Dai 1997 (Decapoda: Brachyura: Potamoidea)

Abstract We report the complete mitochondrial genome of Bottapotamon lingchuanense for the first time, which is found to be 17,612 base pairs in length, and contains 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA), and 1 non-coding AT-rich region known as the D-loop. In addition, the mitogenome has 17 intergenic regions ranging from 1 to 1512 bp in length. The mitochondrial genome of B. lingchuanense is the first mitochondrial genome under the genus Bottapotamon, providing DNA data for species identification, enriching the species diversity of Brachyura. The maximum-likelihood (ML) tree and Bayesian inference (BI) tree based on the 13 PCGs of mitochondrial genome of Brachyura species showed similar topologies with high confidence, and the analysis results were consistent with the current mainstream classification system. The results indicating that B. lingchuanense is closely related to Neilupotamon sinense, Sinopotamon, and Tenuilapotamon, and it is likely to be derived from them.


Brachyuran; mitochondrial genome; Bottapotamon lingchuanense; phylogenetics
The Bottapotamon is a genus of freshwater crab that is unique to mainland China. Up to now, there are eight species under the genus (Bott 1967;Dai 1999;Gao et al. 2019). In mainland China, the species under the Bottapotamon are mainly distributed within the area of the Wuyi Mountain Range, and only the species of Bottapotamon lingchuanense have been isolated in the Nanling Mountain Range (T€ urkay and Dai 1997). The terrain of the habitat of B. lingchuanense is geologically stable, as there is hardly affected by the Cenozoic orogeny (Gao et al. 2019). Therefore, we believe that the distribution of B. lingchuanense in mainland China is caused by the emergence of the mountains. At present, there is no mitochondrial genome data of the species under the genus Bottapotamon in GenBank. Here we present the first mitogenome for the genus Bottapotamon by sequencing the mitochondrial genome of the freshwater crab B. lingchuanense, which has a new mitogenome protein-coding gene order for the Decapoda, aiming to explore the evolutionary relationship within the genus Bottapotamon.
A male adult specimen of B. lingchuanense was collected from Yuanpu Village, Guanyin Township, Gongcheng County, Guilin City, Guangxi Province, China ( ÃÃ latitude ÃÃ 110.9598 and ÃÃ longitude ÃÃ 25.1634) in 2017. The sample is B. lingchuanense, which was identified by morphological methods. The specimen was deposited at the Laboratory Specimen Library of Freshwater Crustacean Decapoda & Paragonimus, School of Basic Medical Sciences, Nanchang University, Nanchang, Jiangxi, PR China & National Parasite Germplasm Resources Specimen Library of China (Jie-xin Zou jxzou@ncu. edu.cn) under the voucher number NCUMCP4076. The sample was fixed in ethanol and then stored at À20 C before sequence analyses. Genomic DNA extraction, sequencing, gene annotation was performed referred to the ways routinely used before (Plazzi et al. 2013).
The complete mitogenome of B. lingchuanense is 17,612 bp in length (GenBank accession number: MN117717) with high A þ T bias (72.33%), containing 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA), and a non-coding AT-rich region and known as the control region. Not as usual, in B. lingchuanense, we can only clearly annotate 20 of the 22 tRNA genes, the positions, and structures of tRNA-Ile and tRNA-Met are slightly abnormal. By aligning it with genetic sequences of near-source species, we can basically confirm that the positions of tRNA-Ile and tRNA-Met should be located in the intergenic region of 149 bp between 12 S rRNA and ND2, and the first 66 bp area is determined as a special tRNA-Ile whose anti-codon is also converted from the usual GAT to AAT. However, the tRNAscan-SE software could not predict the secondary structure of the tRNA, and it could not be spliced into a regular shape by artificial folding neither. The post-83 bp area is the position of the putative tRNA-Met. Compared with the general tRNA-Met (about 70 bp), it is obvious that the length of tRNA-Met in B. lingchuanense is markedly longer. It is suspected that the deletion or replication of the base caused the abnormal of the tRNA genes, which may be caused by the mtDNA copy base deletion mutation.
Using the 48 Brachyura species published by GenBank and the 13 PCGs of mitochondrial genome of B. lingchuanense obtained in this study for phylogenetic analysis, Kiwa tyleri was selected as the outgroup, building maximum-likelihood (ML) and Bayesian inference (BI) trees, respectively. The ML tree and the BI tree obtained by the phylogenetic analysis showed similar topological structure and supported by a high degree of confidence (Figure 1). The results of phylogenetic analysis showed that, consistent with the results of morphological classification analysis, B. lingchuanense with unique gene arrangement and Neilupotamon sinense were clustered together, and they were combined with Sinopotamon and Tenuilapotamon. The composed clades form sister clades, indicating that B. lingchuanense is closely related to Neilupotamon sinense, Sinopotamon, and Tenuilapotamon, and it is likely to be derived from them. University, Nanchang, Jiangxi, PR China) for assistance with specimen collection and statistical analysis.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by the National Natural Science Foundation of China under Grant number [32060306]; National Parasitic Resources Center under Grant number [NPRC-2019-194-30]; and Student's Platform for Innovation and Entrepreneurship Training Program of Nanchang University under Grant number [20190402290].